7890 n gas chromatograph

The SCFAs content in the cecum and colon was determined by gas chromatography (GC). Briefly, the contents were thawed and 0.5 g of the cecum was weighed in a centrifuge tube. It was added with ultrapure water (W:V, 1.0:2.5), and the tube was vortexed for 3–5 min, and then centrifuged at 5,000 rpm for 10 min. Then, 0.2 mL of metaphosphoric acid deproteinizing solution with 2-ethylbutyric acid (2-EB) was added to 1 mL of the obtained supernatant. The mixture was vortexed and then centrifuged for 30 min at 10,000 rpm and 4°C. Finally, the SCFAs content was determined using an Agilent 7,890 N gas chromatograph. The column length was 30 m, the inner diameter was 0.32 mm, the membrane thickness was 0.5 μm, and the injector and detector temperatures were 260°C and 280°C, respectively. The carrier gas was nitrogen at a flow rate of 2.5 mL/min.

Pyrolysis-gas chromatography–mass spectrometry analysis was conducted for DOM characterization using a multi-shot pyrolyzer (PY-3030D, Frontier Laboratories, Fukushima, Japan) attached to an Agilent 7,890 N gas chromatograph (GC) connected to an Agilent 7000B mass spectrometer (MS). The GC was equipped with an elastic Quartz Capillary Column (HP-5MS, 30 m × 0.25 mm × 0.25 μm inner diameter). DOM-containing extracts were lyophilized using a freeze-dryer (ALPHA1-4/Ldplus, Germany). Lyophilized samples (10 mg) were weighted into a small stainless-steel cup and inserted into a pre-heated furnace. The pyrolysis temperature was set as follows: 50°C for 1 min then rose to 600°C, from 50°C to 250°C at a rate of 50°C min⁻¹ and 250°C to 600°C at 30°C min⁻¹. The GC oven was heated from 40°C to 290°C at 4°C min⁻¹. The MS was operated in electron ionization mode (70 eV, scanning 50–550 m/z) with GC injector at 230°C and ion source temperature at 280°C. The carrier gas was helium (1.2 mL min⁻¹). The relative proportion of each compound was equal to the percentage of the peak area of each product to the total peak area.

Total lipid extracts of VC were transesterified using a method described by Stránský and Jursík [33] . Briefly, lipids were dissolved in chloroform:methanol (2∶3, v/v) in a small glass ampoule. After adding acetyl chloride, the ampoule was sealed and placed in a water bath at 70°C. After 60 min the ampoule was opened, the reaction mixture was neutralized with silver carbonate and injected onto GC column. FAME were analyzed using a 7890N gas chromatograph (Agilent, Santa Clara, CA, USA) coupled to a 5975C quadrupole mass spectrometer and equipped with a fused silica capillary column DB-wax (30 m×0.25 mm, 0.25 µm, J&W 122-7032). The carrier gas was helium at 1.5 mL/min. The injector was held at 250°C and operated with a split ratio of 1∶20; 2 µL of sample solution (chloroform:methanol (2∶3, v/v)) was injected. The temperature program: 140°C (0 min), then 5°C/min to 250°C (50 min); total run time was 72 min. 70 eV EI mass spectra were recorded in the mass range of 25–600 u; 3 min solvent delay was used. Temperatures of the transfer line, ion source and quadrupole were 250°C, 230°C and 150°C, respectively. The chromatographic peaks representing FAME were identified based on the presence of m/z 74 and m/z 87 in their mass spectra. FAME were relatively quantified from their peak areas integrated in the total ion current chromatograms.